PROJECT SUMMARY Apolipoprotein-B (ApoB) has recently emerged as a central mediator of metabolic disease, as this single protein induces inflammation, ER-stress, hyperlipidemia, and atherogenesis, and is tightly linked to the incidence of diabetes, metabolic syndrome, and cardiovascular disease. ApoB therefore represents a uniquely promising therapeutic target for the treatment of some of the most prevalent and deadly diseases worldwide, however it has proven difficult to characterize the many factors involved in ApoB regulation and even more challenging to modulate them pharmaceutically. The current proposal seeks to overcome these hurdles by developing the larval zebrafish as a highly tractable platform to study ApoB biology, which will facilitate not only mechanistic investigation of known modulators of ApoB, but also the discovery of completely novel genes and chemical probes relevant to ApoB homeostasis. To this end, zebrafish have been genetically engineered to produce a luciferase reporter fused to ApoB, and analogous fluorescent reporters are currently being developed. These reporters will enable quantitative detection of ApoB levels, the size distribution of ApoB- containing lipoproteins (the beta-lipoproteins), and the localization of beta-lipoproteins in individual zebrafish larvae, enabling researchers to leverage the many advantages of the zebrafish model system to advance the field of ApoB biology. This reporter line will first be used to execute mechanistic studies of the PLA2G12B gene, a novel regulator of lipoprotein biology with no known function. A series of rescue constructs will be used to identify the key functional domains and residues within the protein, and protein interacting partners of PLA2G12B will be identified through co-immunoprecipitation and mass-spectrometry. Integration of these two lines of evidence, will support either a catalytic, receptor-mediated, or scaffolding role for this mysterious protein and potentially implicate additional novel regulators of lipoprotein metabolism. The ApoB reporter line will also be used to execute a high-throughput phenotypic drug screen to identify novel chemical modulators of ApoB, and use the corresponding drug target information to identify novel genes and pathways involved in ApoB regulation. These studies will provide the field with a powerful new model system, as well as novel genes, mechanisms, and chemical probes to advance our understanding of ApoB biology. The availability of such drugs and drug targets related to ApoB has significant potential for translational impact, as this information serves as an excellent starting point for developing therapies to treat diverse metabolic diseases.